In just one decade, green fluorescent protein has rocketed from complete obscurity to dominate the scene in cell biology as a marker for protein localization and gene expression. However, the use of engineered GFP variants as real-time indicators of biochemical events in vivo could have even greater impact on cell biology. Protein engineering and crystallography will be used to develop and improve fluorescent protein indicators for use as visual reporters of redox events and pH in living cells. Available redox biosensors will be redesigned with goals (1) to extend their range of coverage to the redox potentials that obtain in oxidizing subcellular compartments and (2) to increase their sensitivity toward reactive oxygen species (ROS) that act as transient second messengers. A new class of chimeric FRET-based red-green redox indicator will be developed for use in cell-sorting applications. Collaborators have agreed to test new constructions in yeast, plants and mammalian cells. The mechanisms of redox-sensitive GFPs will be investigated. Fluorescent proteins are suspected to generate low levels of ROS that are toxic to cells under certain conditions. The number and nature of probe-generated ROS will be identified and their effects included in analysis of probe response. Finally, the chromophore of GFP is an example of a hydroxyarene photoacid, compounds that can exhibit excited state acidities similar to mineral acids while having a pKa near neutrality in the ground state. GFP provides a unique biological system for study of excited state proton transfer (ESPT) which, in turn, is central to the mechanism of GFP indicators. In a collaborative effort, atomic models and mechanisms for ESPT pathways will be tested for several examples of indicator GFPs using directed mutagenesis, crystallography and ultrafast time-resolved fluorescence spectroscopy.